PROJECT SUMMARY Diabetic cardiomyopathy is a common yet underestimated cause of heart failure and mortality in patients with diabetes. The underlying basis of this often-fatal syndrome is unknown, although multiple pathways converge on a common denominator - dysfunctional mitochondria. Understanding how damaged mitochondria are removed is critical, as the presence of damaged mitochondria leads to rapid generation of reactive oxygen species which then affect the remaining healthy mitochondria. This leads to wide-spread mitochondrial dysfunction and cell death. Although damaged mitochondria are believed to be degraded by mitochondrial autophagy (a specific form of autophagy), we have recently discovered the existence of a novel endosomal- mediated mitochondrial degradation pathway in cardiomyocytes exposed to hyperglycemia. The suppression of this pathway is potentially linked to increased susceptibility against diabetic cardiomyopathy. This observation was possible due to our ability to generate patient-specific induced pluripotent stem cells (iPSCs) from diabetic patients with (T2DCM) and without (T2D) cardiomyopathy. We found that only T2D cells, but not T2DCM, exhibited increased endosomal-mediated mitochondrial degradation. However, neither the molecular cues regulating this novel pathway, nor the functional significance of this endosomal pathway, is established in cardiomyocytes from a diabetic heart. Thus, the goal of this project is to demonstrate the functional significance of the endosomal-mediated mitochondrial degradation pathway during diabetic cardiomyopathy and to elucidate the underlying mechanisms regulating this pathway. Aim 1 will define the functional role of endosomal-mediated mitochondrial clearance in the diabetic heart by disrupting the function of Rab5 and Rab7, key determinants of the endo-lysosomal system. These studies will employ multiple innovative reagents, including CRISPR-mediated Rab knockout and overactivation iPSC lines and a novel inducible, cardiac-specific Rab7 knockout mouse model, to interrogate the importance of these Rabs in maintaining functional mitochondrial degradation in two distinct diabetic mouse models. Aim 2 will define the role of VPS34/UVRAG in generating phosphatidylinositol 3- phosphate (PI3P) required for endosomal maturation and hence clearance of defective mitochondria. We have supporting data that mTOR is excessively upregulated in selected diabetic patients leading to phosphorylation of UVRAG, impairing its function to form a complex with VPS34 in generating PI3P for proper endosomal maturation and mitochondria degradation. Using both genetic and pharmacological methods, we will demonstrate that maintaining a stable UVRAG/VPS34 complex is a prerequisite for PI3P to mediate conversion of Rab5 into Rab7 for endosomal-mediated mitochondrial degradation. Collectively, these innovative studies will illuminate a novel endosomal-mediated mitochondrial degradation pathway as an important adaptive response in cardiomyocytes when exposed to hyperglycemic stress and pave the way for druggable targets in the future.